Introduction- Coxiella burnetii is the etiologic agent of Q fever, a potential bioweapon and select agent, and one of the most infectious pathogens known. However, there are few reports concerning this obligate intracellular agent's molecular pathogenesis or developmental cycle, and lack of a system for site-directed genetic manipulation has severely hampered research progress. To address this dearth of information and to improve the current state of molecular biology technology, we propose to: 1) examine the expression profiles of small cell variants (SCVs) and large cell variants (LCVs) of the developmental cycle to identify genes and proteins that allow Coxiella to survive for extended periods of time in the environment (SCV stage) and to subsequently grow in the inhospitable confines of the host cell phagolysosome (LCV stage); 2) analyze and compare expression profiles of in vitro versus in vivo-cultivated Coxiella and phase I versus phase II organisms to identify potential virulence determinants, and 3) develop a system of site-directed mutagenesis for this organism to allow for the routine genetic manipulation and future development of an attenuated vaccine strain. These data are expected to provide valuable information on the life cycle of Coxiella and the respective genes and proteins involved, and should lead to strategies for disrupting cellular development to control Q fever. Finally, a system for routine genetic manipulation of Coxiella is an essential molecular biology tool for examining suspected virulence genes and performing molecular Koch's postulates in vivo. Project Interactions- Project 3 (Harmsen) will interact with Project 1 (this study) as it relates to in vivocultivated Coxiella from the mouse model. Projects 4 (Pascual) and 2 (Jutila) will benefit from Project 1 data on stage-specific gene products; SCV-specific genes / antigens are hypothesized to be involved in transmission / early infection and LCV-specific genes / antigens are involved in intracellular replication.